Mammalian target of rapamycin (mTOR), a central component of a signaling pathway that coordinates cell growth and nutrient availability, is a promising target for the treatment of diabetes and obesity. The objective of this application is to determine the mechanism through which mTOR is regulated by Rheb, a small Ras- like GTPase downstream of Tuberous Sclerosis Complex 1 and 2 gene products. The central hypothesis for the proposed research is that Rheb regulates mTOR through targeting PLD2 bound to raptor and phosphorylating raptor. The specific aims to test this hypothesis are the followings: (1) to identify the molecular mechanism by which Rheb regulates the raptor-mTOR complex. The working hypothesis is that Rheb targets PLD2 bound to raptor to stimulate the mTOR kinase activity. The hypothesis is based on the finding that Rheb interacts with PLD2 and raptor and that PLD2 is required for Rheb-mediated activation of mTOR signaling. To test the hypothesis, critical residues in raptor and PLD2 involved in the interaction with Rheb will be determined using protein dissection and mutagenesis approaches combined with immunoprecipitation and RNAi. In vivo and in vitro assay will be conducted to determine the effect of Rheb on PLD2 and mTOR activity and of PLD2 on mTOR activity. A PLD2 fragment and PLD2 and raptor mutants will be used to disrupt the interactions between Rheb, PLD2, and raptor and to determine the roles of the interactions in the regulation of the mTOR kinase activity. (2) To identify the molecular mechanism by which Rheb-mediated raptor phosphorylations regulate mTOR signaling. The working hypothesis is that Rheb induces raptor phosphorylations to regulate mTOR activity and raptor stability. The hypothesis is based on the identification of three phosphorylation sites in raptor that are regulated by Rheb. To test the hypothesis, Rheb- and nutrient-regulated phosphorylation sites in raptor will be extensively searched using mass spectrometry and phospho-peptide mapping assay. Raptor mutants will be generated and used for in vivo and in vitro assay to determine the extent to which the mutated phosphorylation sites are involved in the regulation of mTOR activity and raptor stability. Determining the mechanism underlying the up- and down- regulation of mTOR signaling by Rheb and raptor phosphorylations will be crucial in understanding the negative feedback mechanism of the mTOR pathway that is prevalent in insulin resistant cells.